A close association exists between obesity and non-insulin dependent diabetes mellitus (NIDDM). The nature of this link is unclear, but is hypothesized to depend upon high rates of free fatty acid (FFA) release from enlarged abdominal adipocytes. Abnormal regulation of adipocyte triacylglycerol (TG) synthesis and FFA re-esterification may contribute to increased adipocyte size and FFA release. Preliminary data indicate that substrates and hormones control TG synthesis in a complex, interactive fashion. In addition, it has been demonstrated that FFA re-esterification normally takes place via an extracellular route. The importance of these findings for the control of lipid storage in humans will be explored by in vitro incubations of adipocytes. These cells will be derived from both abdominal and gluteal adipose tissue of normal weight, obese, and reduced obese women with predominantly upper or lower body fat distribution. Comparison of adipocytes from these different groups will allow identification of abnormalities that 1) allow enlarged adipocytes from the obese to release FFA at a high rate compared to normals and 2) cause adipocytes from the reduced obese to store lipid very efficiently. The capacity of adipocytes from the different groups to synthesize TG from glycogen or monoacylglycerol (MG) will be compared by incubating adipocytes in presence or absence of glucose and measuring incorporation of 3H-palmitic acid into TG. The specific contribution of glycogen will be assessed by measuring the relative incorporation of 3H from labelled water and 14C from labelled glucose into TG. The contribution of MG will be assessed by measuring the activity of the enzyme monoacylglycerol acyltransferase in human adipocytes from each subject group. Information from these three experiments will be used in the interpretation of the remaining studies, which include: 1) Comparison of the interaction between medium glucose and FFA concentration and hormone and paracrine stimulation of TG synthesis in adipocytes from all subject groups and 2) Determination of the effects of acute hormonal stimulation on FFA re-esterification, unconfounded by changes in lipolysis. TG synthesis will be measured by conversion of 14C-glucose into lipid, lipolysis by glycerol and FFA release, and FFA re-esterification by both traditional and novel techniques. Finally, the use of fluorescent lipid analogues will be developed to investigate the pathways of lipid metabolism in cultured adipocytes.